hodge test
DESCRIPTION
HODGE TESTTRANSCRIPT
HODGE TESTDr.T.V.Rao MD
Dr.T.V.Rao MD 1
Role Carbapenems
• Carbapenems are often used as antibiotics of last
resort for treating infections due to multidrug-
resistant gram-negative bacilli, because they are
stable even in response to extended spectrum and
AmpC -lactamases. However, gram-negative bacilli
producing the acquired metallo-lactamases (MBLs)
IMP and VIM have been increasingly reported in
Asia and Europe
Dr.T.V.Rao MD 2
Emerging Carbapenems Resistance in
Gram-Negative Bacilli
• Significantly limits treatment options for life-threatening infections
• No new drugs for gram-negative bacilli
• Emerging resistance mechanisms, carbapenemases are mobile,
• Detection of carbapenemases and implementation of infection control practices are necessary to limit spread
Dr.T.V.Rao MD 3
Antibiotic Misuse
• Antibiotic misuse, (sometimes called antibiotic abuseor antibiotic overuse) refers to the misuse and overuse of antibiotics which has serious effects on public health. Antibiotic resistant bacteria is a growing threat and becoming increasingly common. This overuse creates multi-antibiotic resistant life threatening infections by "super bugs”, sometimes out of relatively harmless bacteria. Antibiotic abuse also places the patient at unnecessary risk of adverse effects of antibiotics.
Dr.T.V.Rao MD 4
Bugs to Superbugs.
• Antibiotic resistance
develops through gene
action or plasmid
exchange between
bacteria of the same
species. If a bacterium
carries several resistance
genes, it is called
multiresistant or,
informally, a superbug.
Dr.T.V.Rao MD 5
Carbapenem Resistance:
MechanismsEnterobacteriaceae Cephalosporinase + porin loss
Carbapenemase
P. aeruginosa Porin loss
Up-regulated efflux
Carbapenemase
Acinetobacter spp. Cephalosporinase + porin loss
Carbapenemase
Carbapenemases
Classification Enzyme Most Common Bacteria
Class A KPC, SME,
IMI, NMC,
GES
Enterobacteriaceae(rare reports in P. aeruginosa)
Class B
(metallo-β-lactamse)
IMP, VIM,
GIM, SPM
P. aeruginosa
Enterobacteriacea
Acinetobacter spp.
Class D OXA Acinetobacter spp.
Klebsiella Pneumoniae
Carbapenemase • KPC is a class A β-lactamase
– Confers resistance to all β-lactams including extended-spectrum
cephalosporins and carbapenems
• Occurs in Enterobacteriaceae
– Most commonly in Klebsiella pneumoniae
– Also reported in: K. oxytoca, Citrobacter freundii, Enterobacter spp.,
Escherichia coli, Salmonella spp., Serratia spp.,
• Also reported in Pseudomonas aeruginosa (Columbia)
Dr.T.V.Rao MD 8
KPC’s in Enterobacteriaceae
Species Comments
Klebsiella spp. K. pneumoniae-cause of outbreaks
K. oxytoca-sporadic occurrence
Enterobacter spp.
Sporadic occurrence
Escherichia coli
Salmonella spp.
Citrobacter freundii
Serratia spp.
Pseudomonas aeruginosa – Columbia & Puerto Rico
What Labs Should Do Now
• Look for isolates of Enterobacteriaceae (especially K. pneumoniae), with carbapenem MIC ≥ 2 µg/ml or non susceptible to ertapenem by disk diffusion
• Consider confirmation by Modified Hodge Test
• Alert clinician and infection control practitioner to possibility of mobile carbapenemase in isolate
Dr.T.V.Rao MD 10
Control of Infections With Carbapenem-Resistant or
Carbapenemase-Producing Enterobacteriaceae in Acute
Care Facilities
• A difficulty in detecting CRE is the fact that some
strains that harbor blakpc have minimal inhibitory
concentrations (MICs) that are elevated but still
within the susceptible range for
carbapenems. Because these strains are
susceptible to carbapenems, they are not
identified as potential clinical or infection control
risks using current susceptibility testing guidelines.
Dr.T.V.Rao MD 11
Background to Modified Hodge Test
• KPCs are class A carbapenemases that reside on
transferable plasmids and are capable of inactivating
carbapenems, such as imipenem and meropenem. Since
carbapenems are often used to treat infections caused by
extended-spectrum beta lactamase (ESBL)-producing
Gram-negative bacteria, carbapenemase production in
Enterobacteriaceae can significantly limit treatment
options for life-threatening diseases. KPCs occur most
commonly in Klebsiella pneumoniae but have been seen
in other species of Enterobacteriaceae as well.
Dr.T.V.Rao MD 12
CLSI – Modified Hodge Test
• CLSI published a
recommendation that
carbapenems-susceptible
Enterobacteriaceae
with elevated MICs or
reduced disk diffusion zone
sizes be tested for the
presence of
carbapenemases using the
modified Hodge test (MHT).
Dr.T.V.Rao MD 13
Modified Hodge Test
• The Modified Hodge Test (MHT) detects carbapenemase production in isolates of Enterobacteriaceae. The most common carbapenemase found in Enterobacteriaceae is the Klebsiella pneumoniae carbapenemase (KPC). Other carbapenemase, like the metallo β lactamase (MBL) and the SME-1 in Serratia marcescens, can also produce a positive MHT, but are found infrequently in the United States.
Dr.T.V.Rao MD 14
Purpose of Hodge Test
• Carbapenemase production is detected by the MHT when the test isolate produces the enzyme and allows growth of a carbapenems susceptible strain (E.coli ATCC 25922) towards a carbapenem disk. The result is a characteristic cloverleaf-like indentation
Dr.T.V.Rao MD 15
Modified Hodge Test
• The MHT performed on a
100 mm MHA plate. (1) K.
pneumoniae ATCC BAA
1705, positive result (2) K.
pneumoniaeATCC BAA
1706, negative result; and
(3) a clinical isolate,
positive result312
Dr.T.V.Rao MD 16
Reagents and materials
Reagents
• 5 ml Mueller Hinton broth (MHB) or 0.85% physiological saline
• Mueller Hinton agar (MHA)
• 10 μg meropenem or ertapenem susceptibility disk
• E. coli ATCC 25922: 18–24hr subculture
• Equipment
• Turbidity meter
• 35OC ± 2OC ambiant air incubator
• Supplies
• Sterile cotton-tipped swabs
• 1 ml sterile pipette
• Sterile loop
Dr.T.V.Rao MD 17
Specimen and Quality Control
• Specimen
• Test organisms: 18–24 hr subculture
• Special safety precautions
• Biosaftey Level 2
• Quality control
• Perform quality control of the carbapenem disks according to CLSI guidelines.
• Perform quality control with each run.
• MHT Positive Klebsiella pneumoniae ATCC BAA-1705
• MHT Negative Klebsiella pneumoniae ATCC BAA-1706
Dr.T.V.Rao MD 18
Step 1 and 2
• 1.Prepare a 0.5 McFarland
dilution of the E.coli ATCC
25922 in 5 ml of broth or
saline.
• 2Dilute 1:10 by adding 0.5
ml of the 0.5 McFarland to
4.5 ml of MHB or saline
•
Dr.T.V.Rao MD 19
Step 3 and 4
• Streak a lawn of the 1:10
dilution of E.coli ATCC 25922
to a Mueller Hinton agar
plate and allow to dry 3–5
minutes.
• Place a 10 μg meropenem
or ertapenem susceptibility
disk in the center of the test
area.
•
Dr.T.V.Rao MD 20
Step 5 and 6
• In a straight line, streak test organism from the edge of the disk to the edge of the plate. Up to four organisms can be tested on the same plate with one drug.
• Incubate overnight at 35OC ± 2OC in ambient air for 16–24 hour
•
Dr.T.V.Rao MD 21
Interpretation of MHT
• After 16–24 hours of incubation, examine the plate for a clover leaf-type indentation at the intersection of the test organism and the E. coli
25922, within the zone of inhibition of the carbapenem susceptibility disk.
Dr.T.V.Rao MD 22
MHT – Positive Test
• A positive MHT indicates that this isolate is producing a carbapenemase
• Test has a clover leaf-like indentation of the E.coli
25922 growing along the test organism growth streak within the disk diffusion zone.
Dr.T.V.Rao MD 23
Showing the Results on HMT testing
• A positive test indicates carbapenemase production by the test microorganism. By producing carbapenemase, the test microorganism is able to inactivate the carbapenem that diffuses from the disk after the disk has been placed on the Mueller Hinton Agar. This allows carbapenem susceptible E. coli ATCC® 25922™* to grow toward the disk
Dr.T.V.Rao MD 24
MHT – Negative Test
• A negative MHT indicates
that this isolate is not
producing a
carbapenemase
• Test has no growth of the
E.coli 25922 along the test
organism growth streak
within the disc diffusion.
Dr.T.V.Rao MD 25
Showing Positive and Negative isolates
by HMT
• Isolates A and C are
negative for KPC. Isolates
B and D are positive for
KPC as indicated by arcing
growth of the
carbapenem-sensitive E
coli along the clinical KPC
isolates toward the
carbapenem disk.
Dr.T.V.Rao MD 26
What Acquired metallo—lactamases do
(MBL)
• The MBLs efficiently hydrolyze all -lactams, except for aztreonam, in vitro . Therefore, detection of MBL-producing gram-negative bacilli is crucial for the optimal treatment of patients and to control the spread of resistance
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Lee et al - reports Carbapenemase
detection methods• Lee et al. have reported that
the Hodge test can be used to screen carbapenemase-producing gram-negative bacilli and that the imipenem (IPM)-EDTA double-disk synergy test (DDST) can distinguish MBL-producing from MBL-nonproducing gram-negative bacilli
Dr.T.V.Rao MD 28
Phenotypic detection with Hodge test a
Minimal requirement
• Carbapenem resistance and carbapenemase production conferred by blaNDM-1 is detected reliably with phenotypic testing methods currently recommended by the Clinical and Laboratory Standards Institute , including disk diffusion testing and the modified Hodge test
Dr.T.V.Rao MD 29
Modified Hodge Test
Lawn of E. coli ATCC 25922
1:10 dilution of a
0.5 McFarland suspension
Imipenem disk
Test isolates
Described by Lee et al. CMI, 7, 88-102. 2001.Dr.T.V.Rao MD 30
Modified Hodge Test
• Preliminary results suggest that any of the three carbapenem disks work in the Modified Hodge Test
Dr.T.V.Rao MD 31
What Labs Should Do Now
• Look for isolates of Enterobacteriaceae (especially K.
pneumoniae), with carbapenem MIC ≥ 2 µg/ml or non susceptible to ertapenem by disk diffusion
• Consider confirmation by Modified Hodge Test
• Can submit initial isolate to CDC via NJ State Lab for confirmation by blaKPC PCR if KPC-producers not previously identified in hospital’s isolate population
• Alert clinician and infection control practitioner to possibility of mobile carbapenemase in isolate
Dr.T.V.Rao MD 32
Are we losing the Value of
Carbapenems• Carbapenems are the only
antibiotics reliably active against many otherwise multi-resistant gram-negative opportunist bacteria, particularly those with extended-spectrum beta-lactamases (ESBLs) The growing emergence and diversity of carbapenemase producing strains is therefore a major concern.
Dr.T.V.Rao MD 33
CDC reports the new genetic
mechanisms• The isolate, Klebseilla pneumoniae 05-506, was
shown to possess a metallo-beta-lactamase (MBL) but was negative for previously known MBL genes. Gene libraries and amplification of class 1 integrons revealed three resistance-conferring regions; the first
contained bla(CMY-4) flanked by ISEcP1 and blc. The second region of 4.8 kb contained a complex class 1 integron with the gene cassettes arr-2, a new erythromycin esterase gene; ereC; aadA1; and cmlA7
Dr.T.V.Rao MD 34
How we can Improve Hodge Test
• The Hodge test is a simple method for screening
MBL-producing isolates, but occasional isolates
show false-negative results. The test can be
improved by using an IPM disk to which 10 l of 50
mM zinc sulfate (140 g/disk) has been added
(Table 3) or by using Mueller-Hinton agar to which
zinc sulfate has been added to a final
concentration of 70 g/m
Dr.T.V.Rao MD 35
Genetic origin of the NDM-1
• An intact ISCR1 element was shown to be downstream from the qac/sul genes. The third region consisted of a new MBL gene, designated bla(NDM-1), flanked on one side by K. pneumoniae DNA and a truncated IS26 element on its other side. The last two regions lie adjacent to one another, and all three regions are found on a 180-kb region that is easily transferable to recipient strains and that confers resistance to all antibiotics except fluoroquinolones and colistin. NDM-1 shares very little identity with other MBLs, with the most similar MBLs being VIM-1/VIM-2, with which it has only 32.4% identity.
Dr.T.V.Rao MD 36
Molecular configuration of NDM-1
• NDM-1 also has an additional insert between
positions 162 and 166 not present in other
MBLs. NDM-1 has a molecular mass of 28
kDa, is monomeric, and can hydrolyze all
beta-lactams except aztreonam. Compared
to VIM-2, NDM-1 displays tighter binding to
most Cephalosporins.
Dr.T.V.Rao MD 37
NDM genetic coding differs from other
recent isolates
• Compared to VIM-2, NDM-1 displays tighter binding to most cephalosporins, in particular, cefuroxime, cefotaxime, and cephalothin (cefalotin), and also to the penicillins. NDM-1 does not bind to the carbapenems as tightly as IMP-1 or VIM-2 and turns over the carbapenems at a rate similar to that of VIM-2. In addition to K. pneumoniae 05-506, bla(NDM-1) was found on a 140-kb plasmid in an Escherichia coli strain isolated from the patient's feces, inferring the possibility of in vivo conjugation
Dr.T.V.Rao MD 38
Phenotypic detection with Hodge test a Minimal
requirement
• Carbapenem resistance and carbapenemase production conferred by blaNDM-1 is detected reliably with phenotypic testing methods currently recommended by the Clinical and Laboratory Standards Institute , including disk diffusion testing and the modified Hodge test
Dr.T.V.Rao MD 39
Created by Dr.T.V.Rao MD for “ e” learning resources for Medical
Microbiologists in Developing Countries
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